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Abstract

Background

Resistance rates to polymyxin B in surveillance studies have been very low despite
its increasing use worldwide as the last resort therapy for multidrug-resistant Gram-negative
bacilli. However, two other resistance phenotypes, hetero- and adaptive resistance,
have been reported to polymyxin. We aimed to investigate the presence of polymyxin
B hetero- and adaptive resistance and evaluate its stability in carbapenem-resistant
Acinetobacter baumannii (CRAB) clinical isolates.

Methods

CRAB isolates were recovered from hospitalized patients at three Brazilian hospitals.
Hetero-resistance was determined by population analysis profile (PAP). Adaptive resistance
was evaluated after serial daily passages of isolates in Luria-Bertani broth containing
increasing polymyxin B concentrations. MICs of polymyxin B of colonies growing at
the highest polymyxin B concentration were further determined after daily sub-cultured
in antibiotic-free medium and after storage at −80°C, in some selected isolates.

Results

Eighty OXA-23-producing CRAB isolates were typed resulting in 15 distinct clones.
Twenty-nine randomly selected isolates (at least one from each clone) were selected
for hetero- resistance evaluation: 26 (90%) presented growth of subpopulations with
higher polymyxin B MIC than the original one in PAP. No isolate has grown at polymyxin
B concentrations higher than 2 mg/L. Polymyxin B MICs of subpopulations remained higher
than the original population after daily passages on antibiotic-free medium but returned
to the same or similar levels after storage. Twenty-two of the 29 isolates (at least
one from each clone) were evaluated for adaptive resistance: 12 (55%) presented growth
in plates containing 64 mg/L of polymyxin B. Polymyxin B MICs decreased after daily
passages on antibiotic-free medium and returned to the same levels after storage.

Conclusions

The presence of subpopulations with higher polymyxin B MIC was extremely common and
high-level adaptive resistance was very frequent in CRAB isolates.

Keywords:

Background

The increasing worldwide prevalence of multi-drug resistant, Acinetobacter baumannii, a major nosocomial pathogen, particularly carbapenem-resistant strains, is of great
concern, since treatment becomes restricted to very few options [1]. Polymyxins, both B and E (colistin), are “old” polypeptide antibiotics that re-emerged
in clinical practice as the last resort therapy against multidrug-resistant Gram-negative
bacteria; many, including A. baumannii, are only susceptible to these drugs [2]. Although resistance rates to polymyxins in surveillance studies fortunately remain
very low [3], two relatively poorly understood resistance phenotypes, hetero- and adaptive resistance,
have been reported in these drugs [4,5].

The term hetero-resistance refers to a phenotype characterized by the presence of
different (drug-resistant and –susceptible) populations in a single clinical specimen
or isolate [6], while adaptive resistance describes an autoregulated phenomenon characterized by
rapid induction of resistance in the presence of drug and reversal to the susceptible
phenotype in its absence [7].

Hetero-resistance has been recently described for colistin in some carbapenem-resistant
A. baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae isolates [8-10], and other studies have demonstrated the presence of adaptive resistance to polymyxins,
mainly in P. aeruginosa[7]. No study so far has neither investigated the presence of hetero-resistance for polymyxin
B, the frequency of adaptive resistance in carbapenem-resistant A. baumannii (CRAB) isolates, nor assessed the presence of these distinct phenomena in the same
isolates. The aim of this study was to assess the occurrence of these phenomena and
evaluate its stability in CRAB clinical isolates.

Methods

Bacterial strains

CRAB isolates were selected from a total of 132 Acinetobacter spp. isolates (one isolate by patient) consecutively recovered from patients admitted
to three tertiary-care hospitals from Porto Alegre, Brazil, from March to December
2011. Isolates were identified by Vitek 2 system. The following carbapenemase-encoding
genes were examined by multiplex PCR: blaOXA-23, blaOXA-24, blaOXA-51, blaOXA-58 and blaOXA-143 genes [11]. A. baumannii species was confirmed by the presence of the intrinsic blaOXA-51 gene [12].

MICs for polymyxin B, imipenem and meropenem were determined by broth microdilution
and interpreted according to CLSI guidelines [13]. Pseudomonas aeruginosa ATCC 27853 was included as quality control in all tests.

CRAB isolates were submitted to molecular typing by ApaI DNA macrorestriction followed by PFGE [14]. Results were interpreted using a dendrogram constructed using the band-based Dice
coefficient method, and, for the purpose of this study, isolates with >90% were considered
a clone.

Hetero-resistance

Hetero-resistance in selected CRAB isolates was determined by population analysis
profile (PAP). Briefly, solutions containing seven distinct bacterial inoculum, ranging
from 108 (0.5 McFarland standard) to 102 CFU/ml were prepared to facilitate bacterial counting in each plate. A 20 μL aliquot
of each solution was spread on Mueller-Hinton agar plates containing 0, 0.5, 1, 2,
3, 4 and 6 mg/L of polymyxin B. Colonies were counted after 48 h of incubation at
35°C. The limit of counting was 20 CFU/ml. The frequency of hetero-resistant subpopulations
at the highest drug concentration was calculated by dividing the number of colonies
grown on antibiotic-containing plates by the colony counts from the same bacterial
inoculum plated onto antibiotic-free plates [9]. MICs of polymyxin B of these subpopulations growing at the highest polymyxin B concentration
were determined after daily sub-cultured in antibiotic-free medium for 4 days and
after 75 days storage at −80°C, in some selected isolates.

Adaptive resistance

Isolates were submitted to serial daily passages in freshly prepared Luria-Bertani
(LB) broth containing increasing polymyxin B concentrations of 0.25 to 64 mg/L for
a total of nine days (adapted from Fernandez et al. [15]. MICs of polymyxin B of colonies growing at the highest polymyxin B concentration
were also determined after daily subculture in antibiotic-free medium for 3 days and
after 60 days storage at −80°C, in some isolates.

Results

Of the 132 Acinetobacter spp., 124 were confirmed as A. baumannii by the presence of blaOXA-51 gene, and 89 (71.7%) of these were CRAB isolates. All CRAB isolates were positive
for blaOXA-23 and no product of amplification was detected for the other carbapenemase-encoding
genes. Of these, 80 were typed, resulting in 15 distinct clones. MIC50 for both imipenem and meropenem were 64 mg/L and 32 mg/L and MIC90 were 128 mg/L and 64 mg/L, respectively. MIC of polymyxin B ranged from ≤0.125 mg/L
to ≥64 mg/L. Twenty-nine randomly selected isolates (at least one from each clone)
were selected for hetero-resistance evaluation.

PAP revealed the growth of subpopulation with 2- to at least 4 fold dilutions higher
polymyxin B MIC than the original population in 26 (90%) of 29 isolates, including
at least one isolate representative of each clone (Table 1). No isolate has grown at polymyxin B concentrations higher than 2 mg/L. The proportions
of higher MIC subpopulations ranged from 2.5 × 10-7 to 6.2 × 10-4. MICs of polymyxin B of the 26 “higher MIC” subpopulations remained higher than the
original population MIC after daily passages on polymyxin B-free medium (Table 1). After storage, MIC for polymyxin B among 17 selected subpopulations with higher
MIC returned to levels similar to the original population, with most presenting exactly
the same MIC of the original population.

Twenty-two of the 29 isolate (at least one from each clone) were evaluated for adaptive
resistance. In twelve isolates, growth was observed in plates containing 64 mg/L of
polymyxin B (Table 2). After daily passages on polymyxin B-free medium for 3 days the MIC of isolates
growing at 64 mg/L remained the same for two isolates and decreased 1- to 2-fold dilutions
for the other ten (Table 2). Polymyxin B MICs after 60 day storage (performed in four isolates) were exactly
the same of the MIC of baseline.

Discussion

Our study for the first time investigated the presence of hetero- and adaptive resistance
to polymyxin B in unrelated OXA-23-producing CRAB isolates. Additionally, the stability
of these phenomena was evaluated in two distinct conditions. Since the susceptibility
breakpoint for polymyxin B according to CLSI is 2 mg/L [13], real hetero-resistance for polymyxin B was not found in any isolate, differently
from previous studies with colistin [9,16,17]. However, the presence of “higher MIC” subpopulations, within the susceptibility
range, was detected in 90% of tested isolates, including at least one isolate representative
of each of the 15 clones. These “higher MIC” subpopulations presented MICs 2- to at
least 4-fold dilutions higher than the original population.

The presence of adaptive resistance to polymyxin B was shown in 55% of 22 tested isolates
(present in 7 of 15 clones), all demonstrating high-level resistance to polymyxin
B (MIC = 64 mg/L). Although some molecular mechanisms of adaptive resistance to polymyxins,
such as mutations in pmrCAB and lpxA gene in A. baumannii[18,19] and PhoP-PhoQ, PmrA-PmrB and recently ParR-ParS in P. aeruginosa[15], have been characterized, the presence of this resistance phenotype has not been
systematically evaluated. Thus, our study further suggests that adaptative resistance
might be most common than possibly expected, at least in CRAB, since approximately
half of tested clones showed such adaptive phenotype. Indeed, the frequency might
be even higher if the agar plate with the lowest polymyxin B concentration had <0.25
mg/L of the drug. Although seven isolates with MIC ≤0.125 mg/L still have growth on
these plates, these concentrations may have inhibited the growth of other eight isolates.

The present study also showed that the MIC of the “higher MIC” subpopulations remained
stable after 4-days into antimicrobial-free medium, but returns to the MIC of the
original population after storage at −80°C, suggesting that it might involve some
molecular basis also associated with an unstable phenotype. As expected, since without
the drug-sustaining effect the adaptive resistance is unstable, the MICs of resistant
isolates selected in the adaptive resistance experiment decreased 1- to 2-fold dilutions
after serial passage into antimicrobial-free medium and all tested isolates returns
to the baseline level after the storage at −80°C.

Only one isolate that has presented adaptive resistance has not presented “higher
MIC” subpopulation in PAP. It belongs to the clone A, which has other three isolates
tested in both experiments, all showing the presence of both phenomena. It is also
interesting that these latter three isolates were identical by typing while the former
showed 92% of similarity with these latter ones (data not shown). Another isolate
has neither presented “higher MIC” subpopulation nor adaptive resistance and belongs
to a clone with two representative isolates among the 80 CRAB typed in this study.

Unfortunately, we were not able to determine the molecular determinants of these phenotypes
in this study. We also could not determine if the absence of real hetero-resistance
(i.e. presence of subpopulations with MICs higher than the susceptibility breakpoint)
was a specific characteristic of polymyxin B, and would occur with colistin, or “higher
MIC” subpopulations within the susceptibility range was only detected, instead of
subpopulations with “resistance MICs” because the baseline MIC of half of the tested
isolates were very low (≤0.125 mg/L).

In summary, our study showed that the presence of “higher MIC” subpopulations in CRAB
isolates was extremely common. Additionally, high-level adaptive resistance was also
very frequent. The clinical significance of each phenomenon should be further investigated,
since both may potentially affect the outcomes of patients on therapy with polymyxins.

Competing interests

APZ has received consultancy fees from Pfizer, Eurofarma and Forest Laboratories.
All other authors: none to declare.

Authors’ contributions

JB was responsible for performance of the experiments, data interpretation and drafting
the manuscript; BLH performed the experiments and contributed to manuscript draft;
AFM and ALB contributed in the experiments, data interpretation and manuscript draft;
and APZ conceived the study, contributed in data interpretation, drafting and reviewing
of the manuscript. All authors read and approved the final manuscript.

Acknowledgements

This work was supported by Fundo de Incentivo à Pesquisa e Eventos do Hospital de
Clínicas de Porto Alegre (12–0010) and Fundação de Amparo à Pesquisa do Estado do
Rio Grande do Sul (11/0898-3), Brazil. A. P. Z. and A.L.B. are research fellows from
the National Council for Scientific and Technological Development, Ministry of Science
and Technology, Brazil.